The long-term objective of this study is to determine how increased levels of native proteolipid protein (PLP) causes oligodendrocyte death, myelin degradation, and neuronal death. Based upon preliminary data, we hypothesize that insertion of PLP into mitochondria initiates a cascade of events resulting in a compromised mitochondrial membrane potential and decreases in oxidative phosphorylation that lead downstream to oligodendrocyte death, myelin instability, activation of microglia, and increased production of cytokines/chemokines. Increased levels of PLP may be caused by duplications of the native proteolipid protein (PLP1) gene as happens in the human disease Pelizaeus-Merzbacher Disease or by increased expression levels of Plp1mRNA and PLP in normal animals during the peak period of myelination and in aging. The importance of understanding the mechanisms involved in disease progression is that specific drugs could be used to treat PMD patients which are currently available to block upstream and downstream targets of increased levels of PLP. The aim of the present study is to investigate the cellular and molecular sequence of events that cause myelin/oligodendrocyte (Olg) and axonal/neuronal deficits in animal models of PMD, particularly in animals with duplications of the wtPlp1 gene. Mutations of the human PLP1 gene and non-human proteolipid protein gene (Plp1) cause severe motor and cognitive deficits that are associated with shortened lifespan. The histological hallmark of PMD and animal models of PMD are combinations of demyelination or hypomyelination or dysmyelination. Axonal degeneration is also characteristic of most PLP1/Plp1 mutations and is an established factor that contributes to neurological disability. However, no studies have examined how increased levels of PLP leads to microglial activation and neuronal degeneration. This proposal investigates the novel hypothesis that insertion of PLP into mitochondria initiates a cascade of events that causes both autocrine abnormalities in Olgs and paracrine changes in microglia and neurons. Following PLP's insertion into mitochondria, ATP and mitochondrial membrane potentials dramatically decrease. These metabolic changes cause (1) In vitro and in vivo, acidification of media and extracellular fluid (ECF), (2) activation of microglia, and (3) up-regulation of cytokines. We hypothesize that these autocrine and paracrine events contribute to both axonal degeneration and myelin instability. We will test this hypothesis in vivo and in vitro by preventing the insertion of PLP into mitochondria and shifting its transport to myelin. In tissu culture, we made mutant constructs, that when transfected into cells, prevents PLP from trafficking to mitochondria. These tissue culture studies have provided us with the basic data to test our hypothesis. We also made new transgenic mouse lines that prevent PLP's insertion into mitochondria. With these in vivo and in vitro tools, we will be able to determine whether insertion of PLP into mitochondria causes neural abnormalities. PUBLIC HEALTH RELEVANCE: Pelizaeus-Merzbacher Disease (PMD) is an X-linked disorder due to mutations in the proteolipid protein1 (PLP1) gene. PLP1 mutations cause severe motor and cognitive deficits that are associated with shortened lifespan. Duplications of the wild-type (wt) PLP1 gene account for 70% of human PLP1 mutations. The histological hallmark of PMD and animal models (Plp1 mutations) of PMD are combinations of demyelination or hypomyelination or dysmyelination. Axonal degeneration is also characteristic of most PLP1/Plp1 mutations, and is an established factor that contributes to neurological disability. Understanding the molecular and cellular sequence of events that lead to a clinical phenotype is essential to developing therapies for the treatment of PMD. This proposal investigates the novel hypothesis that insertion of proteolipid protein (PLP) into mitochondria initiates a cascade of events that causes both autocrine abnormalities in oligodendrocytes and paracrine changes in microglia and neurons. These events cause (1) acidification of media and extracellular fluid (ECF), (2) activation of microglia, and (3) up- regulation of pro- and anti-inflammatory cytokines. We hypothesize that these autocrine and paracrine events contribute to both axonal degeneration and myelin instability.